INTRODUCTION

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Induction of secretory immunoglobulin A (IgA) responses at the mucosal surfaces (e.g., of the gastrointestinal, respiratory, and genital tracts) is considered to be important for protection against invasive pathogens which colonize the mucosae and secrete harmful toxins (18). In principle, stimulation of the common mucosal immune system by oral immunization with soluble protein immunogens can result in IgA antibodies in various mucosal secretions. However, not only does this require the administration of large and repeated doses of antigen but also the resulting antibody responses are, at best, modest and of short duration, primarily due to the denaturation of antigens by gastric acid or proteolysis by digestive enzymes. One strategy to help prevent the breakdown of orally administered protein antigens involves incorporation of vaccine proteins into particulate antigen delivery systems, such as liposomes or biodegradable microspheres (20). These particles may also serve as depots which prolong the antigenic stimulation by slowly releasing encapsulated antigen.

Liposomes are bilayered phospholipid membrane vesicles that have attracted considerable interest as mucosal delivery systems (8, 14, 21, 24, 26). Following oral administration, the portal of liposome entry into the gut-associated lymphoid tissue (GALT) is believed to be via the M cells of the Peyer's patches. Indeed, liposomes have been visualized in endosomes in M cells and appear to be transported to the underlying lymphoid tissue (1, 23). Despite the use of this promising mucosal vaccination strategy, effective immune responses are not always accomplished. An important obstacle appears to be inefficient uptake by the GALT. This may be partly due to the liposomes getting trapped in the mucous layer that coats the mucosal surfaces and thus failing to reach the mucosal epithelium and consequently the underlying mucosal inductive sites. In general, liposomes may attach to cell surfaces nonspecifically, i.e., electrostatically or hydrophobically, or they may be modified to attach specifically, i.e., via a surface ligand linked to the liposomal membrane which is recognized by a cell surface receptor. For enhanced liposome uptake and augmented mucosal IgA antibody responses, it has been proposed that these particles should be relatively small, to overcome the molecular barrier imposed by the M-cell glycocalyx, and coated with a ligand the receptor of which is expressed by the M cells (7). Under these conditions, the liposome-cell interaction could lead to receptor-mediated endocytosis. However, there is little information regarding the apical membrane molecules that might serve as potential receptors on the M cells. Although several lectins recognize M-cell surface molecules, lectin-targeted particulate systems may be bound and trapped by secreted mucins (22). An alternative ligand that is not bound by mucins is the nontoxic B subunit of cholera toxin (CTB), which has a high affinity for the G_M1 ganglioside, a glycolipid receptor present in the membrane of all nucleated cells, including the apical membrane of the epithelial cells in the intestine. CTB has been previously used to target soluble protein antigens to mucosal surfaces, which results in dramatically increased immune responses (4, 19). To take advantage of this CTB property, we have developed a method for the chemical coupling of CTB to liposomes and have shown that it maintains both its antigenic and binding activities (12). This is important since targeting of liposomes to the GALT would require that CTB maintains its G_M1 binding property. Although G_M1 is not an M-cell-specific receptor, particles coated with CTB are expected to be taken up primarily by these cells. Indeed, even though CTB binds to all intestinal epithelial cells, gold particles coated with CTB bind exclusively to the M cells of the Peyer's patches (22). This is probably because the G_M1 receptor is more accessible on the M cells than on neighboring enterocytes, which possess a much thicker glycocalyx (7).

In this study, recombinant CTB (rCTB) was covalently coupled to the outer surface of small unilamellar liposomes to target delivery of incorporated antigens to Peyer's patches and enhance secretory IgA responses. The model protein antigen encapsulated in liposomes was SBR, which is the saliva-binding region of the AgI/II adhesin from the oral pathogen Streptococcus mutans. Our immune response data show that oral administration of rCTB-coated liposomes to mice induces significantly higher mucosal immune responses to the incorporated SBR antigen than those induced by standard liposome carriers, liposomes in which rCTB has been encapsulated, or liposomes coadministered with rCTB, implying that the observed immunoenhancing effect resulted from increased uptake of the liposomes.

(This research was conducted by Evlambia Harokopakis in partial fulfillment of the requirements for a Ph.D. from the University of Alabama at Birmingham.)

	MATERIALS AND METHODS

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Chemical reagents for liposome preparation. Synthetic phospholipids for liposome preparation (i.e., dipalmitoyl-phosphatidylethanolamine [DPPE], distearoyl-phosphatidylcholine [DSPC], and palmitoyloleoyl-phosphatidylcholine [POPC]) and cholesterol were obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) were purchased from Pierce Chemical Co. (Rockford, Ill.). Dithiothreitol, HEPES, L-lysine, and L-cysteine were obtained from Sigma Chemical Co. (St. Louis, Mo.), and Tris was obtained from Fisher Scientific (Fair Lawn, N.J.). Hanks' balanced salt solution (HBSS) was from GIBCO (Grand Island, N.Y.).

Purification of immunogens. SBR was purified from cell lysates of plasmid SBR-transformed Escherichia coli by using metal chelation chromatography on a nickel-charged column (Novagen) as previously described (13). The affinity of the SBR for nickel arises from a six-residue histidine sequence (at its C-terminal end) which was derived from the expression vector. rCTB was purified from ammonium sulfate-precipitated lysates of a recombinant E. coli clone expressing a plasmid that contained the gene for CTB (5), by using galactose affinity chromatography (27). The concentration of these recombinant protein preparations was estimated by the bicinchoninic acid protein determination assay (Pierce) with bovine serum albumin as the standard.

Liposome preparations and estimation of encapsulated or conjugated antigens. Liposomes with rCTB covalently attached to their outer surface were prepared by procedures previously developed in our lab (12). Briefly, a portion of the DPPE constituent of the liposomes was modified by using the heterobifunctional reagent SMCC. The reaction product, DPPE-MCC, was purified and used along with DSPC, POPC, and cholesterol to form a lipid film on the walls of a round-bottom glass flask. The film was hydrated with HEPES containing 265 µg of purified SBR per ml, which resulted in large multilamellar liposomes. Subsequently, small unilamellar liposomes were produced by extruding the large multilamellar liposomes through a 400-nm-pore-size, followed by a 100-nm-pore-size, membrane. Unincorporated SBR was removed by ultracentrifugation. To conjugate rCTB to the SBR-containing liposomes, thiol groups were added to the lysine residues of the rCTB by means of the amine-reactive reagent SPDP, resulting in rCTB-thiopropionate (rCTB-TP). The thiol groups are necessary for reaction with the maleimide group of the DPPE-MCC constituent of the liposomes. rCTB-TP was then reduced by using dithiothreitol, and the reduced protein was incubated at a final concentration of 1 mg/ml with the liposome suspension. The resulting rCTB-linked liposomes were separated from unconjugated protein by ultracentrifugation and resuspended in an equal volume of HBSS containing 1.5% sodium bicarbonate. Finally, a hemagglutination assay using human erythrocytes enriched with G_M1 (Calbiochem-Behring, San Diego, Calif.) was performed to confirm that rCTB was conjugated to liposomes in a biologically active form.

Immunizations. Ten- to 12-week-old BALB/c mice, from a pathogen-free colony, were used in the oral immunization studies, which were performed in accordance with National Institutes of Health guidelines approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. The following groups of five to six mice (groups A to E) were immunized intragastrically by means of a 22-gauge feeding tube (Popper and Sons Inc., Hyde Park, N.Y.) with the indicated preparation in 0.25 ml of HBSS containing 1.5% sodium bicarbonate: group A received liposomes containing buffer only, group B received liposomes containing the streptococcal SBR antigen, group C received liposomes containing SBR and coadministered with rCTB, group D received liposomes containing SBR plus rCTB (i.e., both antigens encapsulated), and group E received liposomes containing SBR and possessing covalently linked rCTB on their surface.

Sampling and quantification of antibody responses. Preimmune samples of serum and secretions were obtained 1 day before immunization (day 0). Subsequent to immunization, collections were made at weeks 3, 9, 11, 26, 28, and 31. Serum was obtained by centrifugation of blood samples collected from the retroorbital plexus with heparinized capillary pipettes. Saliva samples were collected by means of a pipetter fitted with a plastic tip after stimulation of salivary flow by intraperitoneal injection of 5 µg of carbachol (Sigma Chemical Co.). Fecal extracts were prepared by vortexing three fecal pellets from each mouse in 600 µl of extraction buffer (phosphate-buffered saline containing 0.02% azide, 1% bovine serum albumin, 1 mM phenylmethylsulfonyl fluoride, and 5 mM EDTA) (9). For the vaginal washes, 50 µl of sterile phosphate-buffered saline was inserted into and aspirated from the vagina of each mouse three times. This procedure was performed twice for each collection.

Statistical analysis. Results were evaluated by one-way analysis of variance and the Bonferroni multiple-comparison test by using the InStat program (GraphPad Software, San Diego, Calif.) on a Macintosh computer. Differences were considered significant at a P of <0.05. Antibody data were logarithmically transformed to normalize their distribution and homogenize the variances. The data were finally retransformed and presented as geometric means ×/ standard deviations (SD) for ease of interpretation.

	RESULTS

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Amount of liposome-linked rCTB and liposome-encapsulated SBR or rCTB. The amount of rCTB that was covalently bound to the outer surface of the liposomes was estimated to be approximately 7 to 8% of the quantity added (1 mg/ml of liposome suspension), which was similar to the results in our previous report (12). Therefore, the amount of liposome-linked rCTB given per immunization dose was 17 to 19 µg. Based on this determination, the amount of coadministered rCTB (group C) was set at 18 µg/dose. The encapsulating efficiency of the liposomes was about 4 to 5% of that of the added antigen (SBR or rCTB). It was estimated that the liposomes given to groups B to E contained 3 to 4 µg of SBR per dose and the liposomes given to group D contained, in addition to SBR, 13 to 15 µg of rCTB per dose. Therefore, the amount of encapsulated rCTB was roughly comparable to that of linked or coadministered rCTB.

Serum IgG antibody responses. Oral immunization of mice with a single dose of liposomes containing SBR and with rCTB covalently attached to their outer surface resulted in a strong primary serum IgG response to native AgI/II that was significantly higher (P < 0.001) than those induced by the other SBR-containing liposome preparations (Fig. 1A). The response in the former group was maintained at high levels for at least 6 months and was significantly (P < 0.001) enhanced after a single oral boost (week 26). In contrast, low primary responses to AgI/II were induced by the unconjugated liposome preparations, although the responses were augmented following the booster immunizations, especially in the group given SBR-plus-rCTB-containing liposomes. As expected, serum antibodies to CT (Fig. 1B) were induced only by the rCTB-conjugated, rCTB-containing, or rCTB-coadministered liposome preparations. Preimmune serum samples as well as serum from mice given empty liposomes did not show substantial antibody activity to either AgI/II or to CT (<0.5 µg/ml).

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Serum IgG antibody responses to native AgI/II (A) and CT (B) in groups of mice (groups A to E) orally immunized () at weeks 0, 9, and 26 with one dose of the indicated liposome (L) preparations: group A, empty L; group B, L containing SBR (L-SBR); group C, L containing SBR and coadministered with rCTB [(L-SBR) + rCTB]; group D, L containing SBR plus rCTB (L-SBR/rCTB); or group E, L containing SBR and possessing rCTB on their outer surface [(L-SBR)-rCTB]. The last group was not boosted at week 9. Data represent geometric means ×/ SD of five to six mice per group. For clarity, only the upper or lower SD bars are shown.

Salivary IgA antibody responses. A single oral immunization of mice with the rCTB-conjugated and SBR-containing liposomes resulted in the induction of significantly (P < 0.001) higher levels of salivary IgA antibodies to AgI/II than those induced in the other groups (Fig. 2A). Substantial levels of anti-AgI/II antibodies were maintained for at least 6 months when an oral booster immunization augmented the responses to levels similar to those of the primary response. In the other groups, relatively low levels of salivary antibodies were induced after the primary immunization. These responses were not significantly augmented even after two booster doses (week 9 and 26), with the notable exception of the group given liposomes with encapsulated SBR plus rCTB. Salivary IgA antibodies to CT were detected in all mice receiving rCTB-associated liposome preparations, although the primary response was high only in mice immunized with rCTB-conjugated liposomes. Preimmune salivary samples as well as saliva collected at later time points from control groups contained only background antibody activity to AgI/II or CT.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Saliva IgA antibody responses to native AgI/II (A) and CT (B) in groups of mice (groups A to E) orally immunized () at weeks 0, 9, and 26 with one dose of the following liposome (L) preparations: group A, empty L; group B, L containing SBR (L-SBR); group C, L containing SBR and coadministered with rCTB [(L-SBR) + rCTB]; group D, L containing SBR plus rCTB (L-SBR/rCTB); or group E, L containing SBR and possessing rCTB on their outer surface [(L-SBR)-rCTB]. The last group was not boosted at week 9. Data shown are geometric means ×/ SD of five to six mice per group. For clarity, only the upper or lower SD bars are shown.

Vaginal secretion IgA and IgG antibody responses. Vaginal wash samples from mice immunized with the rCTB-conjugated and SBR-containing liposomes demonstrated considerably higher IgA anti-AgI/II antibody levels (P < 0.001) than the other groups 3 weeks following a single oral immunization (Fig. 3A). Six months later, the responses persisted at substantial levels and could be boosted with a single oral dose. Interestingly, liposomes that carried both SBR and rCTB were the only other preparation that could elicit vaginal anti-AgI/II antibodies at levels significantly higher (P < 0.05) than that of the control preparation (empty liposomes), although two booster immunizations were required. Substantial levels of antibodies to CT were elicited in groups receiving soluble or liposome-associated rCTB (Fig. 3B).

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Vaginal IgA antibody responses to native AgI/II (A) and CT (B) in groups of mice (groups A to E) orally immunized () at weeks 0, 9, and 26 with one dose of the following liposome (L) preparations: group A, empty L; group B, L containing SBR (L-SBR); group C, L containing SBR and coadministered with rCTB [(L-SBR) + rCTB]; group D, L containing SBR plus rCTB (L-SBR/rCTB); or group E, L containing SBR and possessing rCTB on their outer surface [(L-SBR)-rCTB]). The last group was not boosted at week 9. Results are shown as geometric means ×/ SD of five to six mice per group. For clarity, only the upper or lower SD bars are shown.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   Vaginal IgG antibody responses to AgI/II, CT, and total vaginal IgG in groups of mice (groups A to E) orally immunized () at weeks 0, 9, and 26 with one dose of the following liposome (L) preparations: group A, empty L; group B, L containing SBR (L-SBR); group C, L containing SBR and coadministered with rCTB [(L-SBR) + rCTB]; group D, L containing SBR plus rCTB (L-SBR/rCTB); or group E, L containing SBR and possessing rCTB on their outer surface [(L-SBR)-rCTB]). The last group was not boosted at week 9. Data are represented as geometric means ×/ SD of five to six mice per group. For clarity, only the upper SD bars are shown.

IgG/IgA antibody ratios in serum and vaginal secretions. In an attempt to address the issue of possible contribution of plasma-derived immunoglobulins to responses in the genital tract secretions, the mean IgG/IgA ratios of anti-AgI/II and anti-CT antibody responses in serum and in vaginal washes were determined. Calculations were performed only for the group given rCTB-conjugated, SBR-containing liposomes, which exhibited high serum and vaginal antibody responses. For both anti-AgI/II and anti-CT responses, the IgG/IgA ratio was about 100 times higher in serum than in vaginal washes. Specifically, for anti-AgI/II responses, the IgG/IgA ratio was 62.2 ± 52.6 in serum as compared to 0.57 ± 0.54 in vaginal secretions (P < 0.05), while for anti-CT responses, the respective ratios were 13.9 ± 6.55 and 0.13 ± 0.08 (P < 0.05). These data suggest that a considerable amount of vaginal IgA must have been produced locally rather than transported to the vaginal secretion by transduction.

Intestinal IgA antibody responses. IgA anti-AgI/II responses were also detected in fecal extracts from the intragastrically immunized mice. Following the primary immunization, significantly higher (P < 0.05) anti-AgI/II immune responses were detected in mice immunized with rCTB-conjugated and SBR-containing liposomes than in other groups, with the exception of the group which received liposomes containing SBR plus rCTB (Fig. 5A). After the booster immunizations (especially the second one at week 26), IgA anti-AgI/II responses were elevated only in mice receiving rCTB-conjugated or rCTB-containing liposomes. Fecal antibodies to CT were induced in all mice receiving soluble or liposome-associated rCTB (Fig. 5B).

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Intestinal IgA antibody responses to AgI/II (A) and CT (B) in groups of mice (groups A to E) orally immunized () at weeks 0, 9, and 26 with one dose of the following liposome (L) preparations: group A, empty L; group B, L containing SBR (L-SBR); group C, L containing SBR and coadministered with rCTB [(L-SBR) + rCTB]; group D, L containing SBR plus rCTB (L-SBR/rCTB); or group E, L containing SBR and possessing rCTB on their outer surface [(L-SBR)-rCTB]). The last group was not boosted at week 9. Data points represent geometric means ×/ SD of five to six mice per group. For clarity, only the upper or lower SD bars are shown.

	DISCUSSION

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Our results indicate that liposomes can be used as effective oral antigen delivery systems when rCTB is covalently attached to their outer surface. High levels of mucosal IgA antibodies against the incorporated SBR antigen were induced after a single oral immunization of rCTB-conjugated liposomes, whereas plain liposomes or other liposome formulations in which rCTB was either encapsulated or simply mixed with these vesicles were significantly less effective despite the use of multiple doses.

Access to and entrance through the M cells overlying the GALT appears to be a desirable feature for mucosal vaccines, and CTB may constitute an effective means for targeting vaccine particles to the GALT. Such a strategy, however, would require that CTB is attached to the particles in a biologically active form, so that it maintains its G_M1 ganglioside binding ability. The coupling strategy used in our experiments preserves this important feature since liposome-bound rCTB was capable of agglutinating G_M1-enriched erythrocytes. Another important consideration is how accessible the G_M1 receptor on the surface of the M cells is to the vaccine particles. Elegant histological studies have shown that the glycocalyx which coats the M cells, although thinner than that of the neighboring enterocytes, can still be an obstacle when trying to target particles to their cell surface (7). Indeed, these investigators demonstrated that particles 1 µm or larger coated with CTB could not adhere to M cells, unlike smaller CTB-coated particles which could readily bind. The size of our liposomes is restricted to around 100 nm as shown by electron microscopy (12). Another design parameter, which was considered important for enhancing the binding interactions of the rCTB-linked liposomes, involved the use of heterobifunctional linkers which would form a long spacer between rCTB and the surface of the liposomes (15). This would further reduce steric hindrance and allow the liposome-linked rCTB molecule to contact its receptor on the M cell. The rCTB-conjugated liposomes were thus expected to gain access to the GALT, and the observed mucosal IgA responses are consistent with efficient liposome uptake.

Once in the GALT, liposomes can be taken up by antigen-presenting cells, resulting in delivery of the immunogen in a concentrated form to these cells. An interesting speculation is that antigen uptake and subsequent processing within the GALT may be enhanced by an interaction between liposomal rCTB and G_M1 on macrophages acting as antigen-presenting cells. These possible properties of the rCTB-conjugated liposomes may account for the adequacy of a single oral dose in generating strong IgA mucosal immune responses to the liposome-incorporated SBR antigen. For example, 3 weeks following a primary single-dose oral immunization, anti-SBR responses in saliva were about 7% of the total IgA and persisted at substantial levels for at least 6 months.

In contrast to rCTB-conjugated SBR-carrying liposomes, the other experimental liposome preparations required one or two booster immunizations to induce anti-SBR responses at levels higher than the background. These liposomes probably rely only on nonspecific hydrophobic interactions for transport into the inductive sites of the intestinal immune system (7, 8), and thus administration of repeated doses is required for induction of antibody responses. Coadministration of rCTB with SBR-containing liposomes did not result in any immunoenhancing effect. This finding was consistent with previous reports in which commercial CTB or rCTB did not act as an adjuvant for oral immunization when coadministered with immunogens (3, 16). However, when rCTB was coencapsulated with SBR in liposomes, serum and mucosal anti-SBR responses were occasionally higher than those induced by the liposomes containing only SBR. It is possible that rCTB can act as a mucosal adjuvant when it is delivered to the GALT with the target immunogen within a vehicle. In this regard, we previously found that anti-SBR responses were higher after immunization with an attenuated Salmonella typhimurium vector coexpressing SBR and CTB than after immunization with a similar clone expressing SBR alone. Immunomodulating functions reported for rCTB, such as enhancement of antigen presentation by macrophages (17) and induction of class II major histocompatibility antigens on B cells (6), may not only contribute to its strong immunogenicity but, since they are antigen nonspecific, may also enhance immune responses to other antigens that happen to be in the same microenvironment with CTB.

The SBR is the adherence domain of AgI/II, a major adhesin implicated in the initial adherence of S. mutans to the salivary pellicle-coated tooth surfaces (2, 10). Recently, we have shown that intranasal immunization with a soluble chimeric protein consisting of the SBR and the A2 and B subunits of CT protects against experimental S. mutans-induced dental caries (11). The rCTB-conjugated liposome strategy induces a higher salivary IgA anti-SBR response than the soluble SBR-CTA2/B chimeric protein does (9), and thus it is similarly expected to confer protection against S. mutans-induced caries. Moreover, the ability of rCTB-conjugated liposomes to induce high levels of specific antibodies in the genital tract after a single oral immunization (specific IgA responses in the vaginal secretions were about 5% of the total IgA) is an important finding since it suggests that this oral vaccination strategy may find application in the prevention of sexually transmitted diseases. Both IgA and IgG antibodies were detected in vaginal secretions, and our results indicate that a significant portion of the IgA antibodies must have been produced locally since the IgG/IgA ratio did not reflect that of the serum.

The impressive systemic and mucosal immune responses generated by a single oral dose of antigen encapsulated in rCTB-conjugated liposomes indicate that targeting of nonliving microparticulate vaccine vectors to GALT via rCTB may be a promising way of circumventing problems associated with oral immunization. The high efficiency of this nonliving vaccine delivery system may be preferred over that of attenuated recombinant bacterial or viral vectors for safety reasons as well as to avoid stimulation of the immune system in response to unrelated antigens carried by the vector.